Different Concentrations Of Artesunate Research Articles

Based on NF-κB and Notch1/Hes1 Signaling Pathways, the Mechanism of Artesunate on Inflammation in Osteoporosis in Ovariectomized Rats was Investigated.

Artesunate (ART) has the potential to modulate the nuclear factor kappa B (NF-κB) and Notch1/Hes1 signaling pathways, which play crucial roles in the pathogenesis of osteoporosis. This study aims to explore whether ART participates in the progression of osteoporosis by regulating these signaling pathways. In the in vitro experiments, we treated bone marrow mesenchymal stem cells (BMSCs) with different concentrations of ART (0, 3, 6, 12 µM) and evaluated osteogenic differentiation using alkaline phosphatase staining (ALP) and alizarin red S staining (ARS) staining. The expression levels of osteocalcin (OCN), RUNT-related transcription factor 2 (RUNX2), osteoprotegerin (OPG), and receptor activator of the nuclear factor kappa ligand (RANKL) were detected by real-time quantitative PCR (RT-qPCR). The effects of ART on NF-κB p65 and Notch1 protein expression were analyzed by Western blot (WB) and immunofluorescence (IF). In the in vivo experiments, a postmenopausal osteoporosis rat model was established via ovariectomy. Bone tissue pathological injury was evaluated using hematoxylin eosin (HE) staining. Serum ALP levels were measured using a kit, bone density was determined by dual-energy X-ray absorptiometry, and serum levels of bone gla protein (BGP), OPG, RANKL, tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), and IL-1β were measured by enzyme-linked immunosorbent assay (ELISA). Additionally, the expression of NF-κB p65 and Notch1 in tissues was assessed by immunohistochemistry. In vitro experiments revealed that compared to the control group, ART dose-dependently promoted BMSCs proliferation and enhanced their osteogenic differentiation capability. The expression of OCN, RUNX2, and OPG significantly increased in the ART-treated group, while RANKL expression decreased significantly (p < 0.05). ART significantly inhibited the expression of NF-κB p65 and Notch1/Hes1 signaling pathway proteins (p < 0.05). Compared to ART treatment alone, combined treatment with ART and phorbol myristate acetate (PMA) or valproic acid (VPA) resulted in increased expression of NF-κB p65 and Notch1 proteins and decreased osteogenic differentiation capability (p < 0.05). In vivo experiments showed that in rats treated with ART, bone damage was significantly reduced, bone density and mineral content were restored considerably, and the expression of inflammatory factors (TNF-α, IL-6, IL-1β) decreased significantly (p < 0.05). Additionally, ART treatment significantly reduced the expression of NF-κB p65 and Notch1 proteins, increased OPG expression, and decreased BGP and RANKL levels (p < 0.05). In summary, ART facilitates the osteogenic differentiation of BMSCs by inhibiting the NF-κB and Notch1/Hes1 signaling pathways, thereby exerting significant protective effects against osteoporosis.

Artesunate regulates the proliferation and differentiation of neural stem cells by activating the JAK‑2/STAT‑3 signaling pathway in ischemic stroke.

Ischemic stroke is one of the most common causes of disability and death globally; therefore, the repair and reconstruction of the central nervous system (CNS) after stroke is very important. Neural stem/progenitor cells (NSPCs) may be the key to cell replacement therapy to treat CNS damage. It has previously been reported that artesunate (ART) is involved in the regulation of the biological functions of NSPCs; however, the mechanism of action of ART remains unclear. In the present study, different concentrations of ART were used to treat NSPCs following oxygen-glucose deprivation (OGD). Cell viability and apoptosis were analyzed using Cell Counting Kit-8 assay and flow cytometry, respectively, whereas immunofluorescence analysis was used to measure the expression levels of the differentiation-related molecule doublecortin (DCX) and proliferating cell nuclear antigen (PCNA). Western blotting was performed to analyze the expression levels of molecules related to the JAK-2/STAT-3 signaling pathway. The present results indicated that treatment with ART following OGD significantly promoted the viability of NSPCs, inhibited the apoptosis of NSPCs, and promoted the expression of PCNA and DCX. Moreover, ART significantly downregulated the protein expression levels of phosphorylated (p)-JAK-2 and p-STAT-3. Furthermore, activation of the JAK-2/STAT-3 signaling pathway and treatment with ART reversed the effects of ART on the proliferation, apoptosis and differentiation of NSPCs. In conclusion, the present data suggested that ART may promote the proliferation and differentiation of NSPCs, and reduce the apoptosis of NSPCs, by inhibiting the JAK-2/STAT-3 signaling pathway. ART may potentially be used for the treatment of ischemic stroke.

Abstract 1119: Non-adherent breast and non-small-cell lung cancer cell cultures as a promising CTCs’ model for evaluation of the anti-tumor effects of artesunate

Abstract Background: Artesunate (AS), besides being widely used as an antimalarial agent, has been also suggested to exert anti-tumor activity in a plethora of cancer types, including breast and lung cancer. Recently, AS has been shown to inhibit the expression of JunB, which is associated with metastatic progression. The aim of this study is to examine the effect of AS on the viability of both adherent and non-adherent (resembling circulating tumor cells; CTCs) breast and lung cancer cell cultures. Methods: Prevention of cell attachment and establishment of the CTCs’ model was achieved via polyHEMA [poly(2-hydroxyethyl methacrylate)] treatment. Viability of adherent and non-adherent MDA-MB-231 (triple negative breast cancer) and H1299 (non-small-cell lung cancer) cells was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide (MTT) assay, 48 h following treatment with different concentrations of AS (3 μM to 100 μΜ) in serum starvation conditions. Cell migration was examined using the wound healing assay. Results: Optimization involving the duration (20 min vs. overnight) and concentration (10 mg/ml) of polyHEMA coating, as well as the duration of AS treatment (24 h vs. 48 h) were performed. 5-fluorouracil was used as a positive control. AS decreased viability of both cell lines in a concentration-dependent manner with comparable IC50, whether being adherent (pIC50 = 4.6 ± 0.1 and 5.1 ± 0.1 for MDA-MB-231 and H1299, respectively; n = 8) or non-adherent (pIC50 = 4.5 ± 0.1 and 4.9 ± 0.1 for MDA-MB-231 and H1299, respectively; n =8). AS (10 μΜ, 48 h) inhibited proliferation of both adherent MDA-MB-231 (34% ± 5, n = 8) and H1299 (57% ± 4, n = 8) cells. Interestingly, the effect of AS on MDA-MB-231 and H1299 non-adherent cultures was less prominent (15% ± 6 and 26% ± 6, P&amp;lt;0.001, respectively; n = 8), demonstrating resistance to AS. Furthermore, AS (10 μM, 48 h) decreased cell migration, an effect which was more pronounced in H1299 cells. Conclusions: Evidently, non-adherent, free-floating cells seem to respond differently to AS compared to their attached counterparts. These results suggest that antitumor agents might exert different effects in CTCs compared to adherent tumor cells. Acknowledgements: This research has been co-financed by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH - CREATE - INNOVATE (project code: T2ΕΔΚ-01562). Citation Format: Evangelia Pantazaka, Dafni Graikioti, Constantinos M. Athanassopoulos, Galatea Kallergi. Non-adherent breast and non-small-cell lung cancer cell cultures as a promising CTCs’ model for evaluation of the anti-tumor effects of artesunate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1119.

Artesunate-induced Cellular Effects Are Mediated by Specific EPH Receptors and Ephrin Ligands in Breast Carcinoma Cells

The aberrant regulation of erythropoietin-producing hepatocellular carcinoma (EPH) receptors and ephrin ligands has been implicated in breast carcinoma, and artesunate has been shown to have anticancer effects. The aim of this study was to characterize the involvement of EPH receptors and ephrin ligands in mediating artesunate (ART)-induced growth suppression of normal breast cells and breast carcinoma cell lines. The normal breast epithelial cells (MCF10A), non-invasive ductal breast carcinoma cells (MCF7), and invasive triple-negative breast carcinoma cells (MDA-MB-231) were grown in the absence or the presence of different concentrations of artesunate. The cells were counted, and total RNA was isolated. The abundance of transcripts corresponding to EPH receptors and ephrin ligands was determined by quantitative polymerase chain reaction. Cell viability was significantly reduced when cells were treated with artesunate, with MDA-MB-231 cells having the highest sensitivity. Artesunate had no significant effect on transcription of EPH/ephrins in MCF10A cells, but markedly increased EPHA8, EPHA10, EPHB6 and ephrin-A2 expression in MCF7 cells, and significantly increased EPHA3 and EPHA10 expression while reducing that of EPHA7 and ephrin-A3 in MDA-MB-231 cells. The relative changes in artesunate-treated MCF7 and MDA-MB-231 cells as compared to similarly treated MCF10A cells allow us to implicate combinatorial expression and receptor interactions for EPH receptor-mediated signal transduction that converges into pathways responsible for cell growth, proliferation, and apoptosis. Specifically, the alterations in EPHA7, EPHA8, EPHA10 and EPHB6 transcripts appear to be important participants in artesunate-mediated cellular effects.

Artesunate attenuates proliferation of epithelial cells by downregulating the NF-κB and AKT signaling pathways in benign mammary gland hyperplasia rats.

BackgroundThe aim of this study was to investigate the effects of artesunate (ART) on breast epithelial cell proliferation in vitro and in vivo.MethodsImmortalized human non-cancer mammary epithelial (MCF-10A) cells were used to determine the effect of ART on estrogen-induced mammary hyperplasia cells. We investigated the effect of ART on the synthesis of cyclooxygenase-2 (COX-2) and proliferating cell nuclear antigen (PCNA) in MCF-10A by treating MCF-10A 36 h with different concentrations of ART (0, 100, 200, 400 µm, n=12/group). We then investigated the effect of ART on estrogen induced COX-2, PCNA, nuclear factor-kappa B (NF-κB), and pNF-κB synthesis by treating MCF-10A with both estrogen and ART (0, 50, 100, 200 µm, n=12/group). A mammary hyperplasia model (MGH) was established in rats. All rats (n=12) were divided into 4 groups [group A: negative control (NC) + Art −; group B: NC + Art +; group C: MGH + Art −; group D: MGH + Art +] by the random number table method and the effects of ART on estradiol-induced mammary hyperplasia, fibrosis, and phosphorylation of AKT and NF-κB were studied by histopathological staining, Masson trichrome staining, immunohistochemistry (IHC), and western blotting.ResultsThe proliferation and inflammation of mammary epithelial cells were blocked by ART (P<0.05). The phosphorylation of NF-κB induced by estradiol in MCF-10A was attenuated by ART (P<0.05). In the rat MGH, ART reduced cell proliferation and fibrosis (P<0.05) and inhibited the phosphorylation of AKT and NF-κB (P<0.05).ConclusionsThe drug ART inhibits estrogen-induced breast hyperplasia by blocking AKT and NFkB phosphorylation.

Artesunate exhibits synergistic anti-cancer effects with cisplatin on lung cancer A549 cells by inhibiting MAPK pathway

BackgroundArtesunate (ART) has been used extensively as anti-malarial drugs worldwide. Besides, it has also been reported to have anti-cancer activities. This study was aimed to explore the anti-cancer activity of ART in combination with cisplatin (CIS) on A549 cells. MethodsCells were cultured with different concentrations of ART and/or CIS for 24, 48, or 72 h to test the anti-proliferative effects by CCK-8 assay. Colony formation assay and EdU staining were also performed. TUNEL staining was used to illustrate the morphologic changes. Cell cycle and apoptosis were determined by flow cytometry assay, and Western blot analysis was conducted to detect the expression of apoptosis- and proliferation-related proteins. Caspase activities were determined by colorimetric assay kit. Moreover, the synergistic effect of ART with CIS in A549 cell xenograft model was also determined. ResultsART significantly inhibited cell proliferation in dose- and time-dependent manners. Collectively, the combination treatment remarkably decreased colony formation rates and increased the rates of TUNEL-positive cells compared with mono-treatment. Mechanistically, the combination treatment influenced the expression of Bcl-2, Bax, p-P53, Caspase-3/7, Caspase-9, CyclinB1, P34, P21, and synergistically regulated the activity of P38/JNK/ERK1/2 MAPK pathway. In mice A549 xenograft tumors, the combination strategy significantly increased the anti-cancer efficacy of ART and CIS alone, consistent with the in vitro observations. ConclusionsART exhibited significant anti-tumor effect on A549 cells and this efficiency could be enhanced by combination with CIS.

Abstract 3805: Artesunate reduces tumor growth in sunitinb-resistant renal cell carcinoma cells

Abstract Introduction: Renal cell carcinoma (RCC) is the most common cancer of the kidney. Due to therapy resistance effect of the approved compounds, like the tyrosine kinase inhibitor (TKI) sunitinib, is limited. Thus, new treatment approaches are urgently needed. Artesunate (ART) is derived from Traditional Chinese Medicine (TCM), showing anti-tumor activity, also in urologic tumors. However, little is known about the efficiency of ART on therapy-resistant RCC. Therefore, we investigated the effect of ART on sunitinib-resistant RCC cell lines. Material and Methods: Parental (=sensitive) and sunitinib-resistant (1 µM) RCC cell lines, KTC-26, A498, and Caki-1, were exposed to ART (1-100 µM) for 24, 48 and 72 hours. ART untreated cells served as controls. Tumor cell growth, proliferation, cell cycling and the expression of cell cycle regulating proteins were then evaluated. Aside, the appearance of apoptotic and necrotic effects was determined. Results: Exposure to different concentrations of ART significantly reduced the tumor cell growth and proliferation of parental and sunitinib-resistant cells in a time- and dose-dependent manner, compared to the controls. At the same time, exposure to ART resulted in a cell cycle arrest in the G0/G1 phase. Notably, this effect was more prominent in the sunitinib-resistant cells. G0/G1 phase elevation was associated with a decrease in the percentage of S and G2/M phase cells. The observed changes in cell growth and cell cycle phases were accompanied by distinct modulations of the cell cycle regulating proteins, indicating for an inhibitory regulation of ART through cell cycle arrest. Indeed, neither necrotic nor apoptotic effects events were detectable after ART treatment, corroborating this assumption. Conclusion: Due to our data we conclude that ART has anti-tumor effects not only in parental but also in sunitinib-resistant RCC cells. Thus, adding ART to the standard therapies might be a promising treatment strategy for patients with advanced RCC. Further investigations are necessary to verify our findings. Citation Format: Eva Juengel, Sascha Markowitsch, Holger H. Erb, Thomas Efferth, Axel Haferkamp. Artesunate reduces tumor growth in sunitinb-resistant renal cell carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3805.

Effects of artesunate combined with bortezomib on apoptosis and autophagy of acute myeloid leukemia cells in vitro and its mechanism

目的探讨青蒿琥酯联合硼替佐米对急性髓系白血病（AML）细胞株MV4-11细胞增殖、凋亡及自噬的影响及其作用机制。方法MTT法检测青蒿琥酯、硼替佐米单用及两药联合对MV4-11细胞的增殖抑制作用，流式细胞术检测细胞凋亡情况，Western blot法检测细胞内Bcl-2家族蛋白（Bcl-2、Mcl-1、Bim、Bax）、cleaved-Caspase-3及自噬相关蛋白LC3B的表达。结果青蒿琥酯呈浓度和时间依赖性抑制MV4-11细胞增殖，作用48 h的IC50为1.44 µg/ml。硼替佐米呈浓度依赖性抑制MV4-11细胞增殖，作用48 h的IC50为8.97 nmol/L。青蒿琥酯（0.75、1.0 µg/ml）与硼替佐米（6、8 nmol/L）联合作用48 h，对MV4-11细胞的增殖抑制率均高于单药组（P值均<0.05），两药相互作用的协同指数（CI）均<1。1.5 µg/ml青蒿琥酯作用MV4-11细胞48 h的凋亡率为（15.27±2.18）%，8 nmol/L硼替佐米作用MV4-11细胞48 h的凋亡率为（19.85±3.23）%，两药联合作用后凋亡率增高至（81.67±5.96）%，显著高于单药组（P值均<0.05）。两药联合作用MV4-11细胞24 h后细胞内cleaved-Caspase-3、促凋亡蛋白Bim及自噬相关蛋白LC3B表达上调，抗凋亡蛋白Bcl-2表达下调。结论青蒿琥酯联合硼替佐米具有协同抑制MV4-11细胞增殖和诱导凋亡及促进自噬的效应，作用机制可能与Bcl-2家族蛋白表达的改变有关。

The therapeutic effect of artesunate on rosacea through the inhibition of the JAK/STAT signaling pathway.

Acne rosacea is a type of chronic dermatosis with the characteristics of erubescence, angiotelectasis and pustule formation. However, current treatment methods are limited due to the side effects. Artesunate demonstrated a promising therapeutic efficacy with a high safety margin. HaCaT cells were treated with antibacterial peptide LL‑37 to simulate rosacea caused by Demodex folliculorum (D. folliculorum) infection. Cell Counting kit 8 and flow cytometry assays were performed to measure cellular proliferation, apoptosis, the stage of the cell cycle and reactive oxygen species generation in order to determine the level of cell damage. Then the damaged cells were treated with different concentrations of artesunate and doxycycline to determine the therapeutic effect of artesunate. Pro‑inflammatory cytokines tumor necrosis factor‑α (TNF‑α), interleukin (IL)‑6, IL‑8 and C‑C motif chemokine 2 (MCP‑1) were measured using an ELISA, while western blotting was used to detect the expression of Janus kinase 2 (JAK2) and signal transducer and transcription activator (STAT3). As a result, LL‑37 treated HaCaT cells decreased in cell viability, had an increased apoptotic rate and cell cycle arrest, indicating that cell damage caused by rosacea was simulated. In addition, upregulated concentrations of the pro‑inflammatory cytokines TNF‑α, IL‑6, IL‑8 and MCP‑1 were attenuated in the artesunate group in a dose‑dependent fashion, indicating the therapeutic effect of artesunate. Furthermore, higher concentrations of artesunate exhibited an improved effect compared with the doxycycline group. In addition, increased expression levels of JAK2 and STAT3 following treatment with LL‑37 suggested that rosacea caused by D. folliculorum infection may lead to inflammation through the JAK/STAT signaling pathway. In conclusion, the potential mechanism by which damage occurs in rosacea was revealed and a promising therapeutic method against rosacea was demonstrated.

Artesunate inhibits proliferation of glioblastoma cells by arresting cell cycle

Glioblastoma is a common brain tumor and the overall survival rate of the patients is very low, so it is an effective way to develop the potential chemotherapy or adjuvant chemotherapy drugs in glioblastoma treatment. As a well-known antimalarial drug, artesunate(ARTs) has clear side effects, and recently it has been reported to have antitumor effects, but rarely reported in glioblastoma. Different concentrations of ARTs were used to treat the glioblastoma cells, and then the inhibitory effect of ARTs on glioblastoma proliferation was detected by MTT assay; Ki67 immunofluorescence assay was used to detect the proliferation of cells; Soft agar experiment was used to explain the clonal formation abilities in vitro; Flow Cytometry was used to detect the cell cycle; and Western blot assay was used to determine the expression of key cell cycle protein. MTT assay results indicated that ARTs-treated glioblastoma cell A172, U251, U87 were significantly inhibited in a time-and-dose dependent manner as compared to the control group(DMSO treatment group). Soft agar experiment showed that ARTs could significantly reduce the clonal formation ability of glioblastoma. Furthermore, Flow cytometry analysis showed that ARTs could obviously increase the cell proportion in G₀/G₁ phase and reduce the cell proportion in S phase. Western blot results showed that the expressions of cell cycle-related proteins CDK2, CDK4, cyclin D1 and cyclin B1 were all obviously down-regulated. Above all, ARTs may inhibit the proliferation of glioblastoma cells by arresting cell cycle in G₀/G₁ phase through down-regulating the expression of CDK2, CDK4, cyclin D1, cyclin B1. These results may not only provide a novel method for rediscovering and reusing ARTs but also provide a new potential drug for treating glioblastoma.

Role of artesunate in TGF‑β1‑induced renal tubular epithelial‑mesenchymal transdifferentiation in NRK‑52E cells.

The implications of epithelial-mesenchymal transdifferentiation (EMT) have extended beyond the confines of renal fibrosis to renal tubulointerstitial fibrosis. It has been proposed that EMT may be one of the mechanisms involved in the pathogenesis of renal fibrosis. However, the underlying mechanisms remain unknown. Transforming growth factor (TGF)-β1 is considered to be an important cytokine which regulates the transdifferentiation of tubular epithelial cells into myofibroblasts in renal tubulointerstitial fibrosis. In the present study, normal rat kidney tubular epithelial cells (NRK-52E) were treated for 48 h with TGF-β1 (5 ng/ml) and different concentrations of artesunate (ART; 0.01, 0.1 and 1 µg/ml). Western blotting, reverse transcription-semi quantitative polymerase chain reaction analysis and immunofluorescence staining were used to evaluate the expression of bone morphogenetic protein (BMP)-7, uterine sensitization-associated gene (USAG)-1, E-cadherin, α-smooth muscle actin (α-SMA) and extracellular matrix collagen type I (Col I) mRNA. ART was able to attenuate renal injury in a unilateral ureteral obstruction model. However, its anti-fibrotic effect remains to be elucidated. In the present study, it was observed that ART was able to ameliorate the TGF-β1-induced alterations in cellular morphology. In addition, ART inhibited the TGF-β1-induced USAG-1 increase and the decrease in BMP-7. Treatment with ART markedly attenuated the TGF-β1-induced upregulation of α-SMA and downregulation of E-cadherin. Additionally, ART was able to significantly attenuate the deposition of interstitial collagens, including Col I. The results of the present study further verified the therapeutic efficacy of ART in TGF-β1-induced renal interstitial fibrosis. These findings indicated that ART may hold the potential to prevent chronic kidney diseases via the suppression of USAG-1 expression or by increasing BMP-7 expression.

Artesunate induces apoptosis via inhibition of STAT3 in THP-1 cells

ObjectiveOur objective was to explore STAT3 expression in patients with acute myeloid leukaemia (AML), assess the anti-proliferative effects of artesunate (ART) on THP-1 cells in vivo and in vitro, and investigate the underlying mechanisms. MethodsIn this study, we examined 30 patients with acute myeloid leukaemia diagnosed in our hospital from January 2015 to January 2016. The 20 control group patients had non-haematological diseases and were hospitalized for the same period. We extracted 2ml bone marrow, separated the mononuclear cells, obtained total proteins, and detected STAT3 protein levels with Western blot analyses. The THP-1 cells were treated with different concentrations of ART(0, 10, 25, 50, 100, 200μM). Then, THP-1 cell viability was detected with CCK-8 assays, apoptosis was measured with flow cytometry, and the STAT3, caspase-3 and caspase-8 protein levels were assessed using Western blot analyses. THP-1 cells in logarithmic growth phase were subcutaneously injected into the necks of 5-week-old nude mice. The control group was subcutaneously injected with 0.1ml PBS. After the nude mouse tumours grew, the mice were divided into the control group and drug intervention groups (ART 100μM group, ART 200μM group). The mice in the intervention groups were intraperitoneally injected with ART, and the control group was injected with the same amount of normal saline. Then, changes in the tumours were observed. After the drug intervention, the total protein was extracted, and STAT3 expression was detected by Western blot analysis. ResultsCompared with the control group, the AML patients had significantly increased STAT3 protein levels (P<0.01). ART significantly inhibited the proliferation of THP-1 cells in a dose-dependent and time-dependent manner. ART also increased THP-1cell apoptosis. After treatment with ART, STAT3 protein was significantly down-regulated, and apoptosis of the cells was induced by the activation of caspase-3 and caspse-8. ConclusionAML patients had higher expression of STAT3 than that of the controls. ART induced apoptosis in THP-1 cells and inhibited the growth of xenografts in nude mice, and we also observed that ART down-regulated the expression of STAT3 and activated the caspase-3 and caspase-8. We speculated that the effect of ART on THP-1 cells may be related to inhibition of STAT3 and activation of caspase3 and caspase-8.

Inhibitory Effect of Artesunate on Growth and Apoptosis of Gastric Cancer Cells

The present study aimed to explore the inhibitory effect of artesunate on the growth and apoptosis of gastric cancer cells, in order to search for high effective and low toxic anti-gastric cancer drugs. Flow cytometry was used to detect the CDC25A protein expression in different gastric diseases. After the treatment of different concentrations of artesunate (0, 30, 60, 120μmol/l) on the gastric cancer cell line SGC-7901cells for 24h, 0μmol/l Art was instead of normal saline as control group, the expression of CDC25A, Bcl-2, Bax, Caspase-3 protein, mitochondrial membrane potential, cell apoptosis and cell cycle were detected by flow cytometry. The CDC25A protein expression in gastric adenocarcinoma was significantly higher than that in normal gastric tissues. After the treatment of different concentrations of Art, the apoptosis rate of SGC-7901cells in Art groups was significantly higher than that in control group. The proliferation index and CDC25A protein of SGC-7901cells in Art groups was significantly lower than that in control group. Compared with control group, the Bcl-2 protein and mitochondrial membrane potential was significantly lower but the Bax and Caspase-3 protein expression level was significantly higher. The high expression of CDC25A protein in gastric adenocarcinoma is involved in the occurrence and development of gastric adenocarcinoma. Artesunate can inhibit the growth of gastric adenocarcinoma cells SGC-7901 and induce the cell apoptosis, the mechanism may be related to the regulation of CDC25A, Bcl-2, Bax, Caspase-3 and mitochondrial membrane potential in SGC-7901cells.

Induction of Apoptosis in Human Breast Cancer MCF-7 Cells by a Semi-Synthetic Derivative of Artemisinin: A Caspase-Related Mechanism.

Background: Artesunate has recently been used in some pharmacological preparation to induce tumor cell apoptosis. The drug is a semi-synthetic derivative of artemisinin, traditionally used for its antimalarial. However, up to now, its anticancer mechanism against different types of tumors is not known. Objectives: The most important purposes of the present research was firstly investigating induction of apoptosis on human breast cancer MCF-7 cells by the drug and, in the second place, introducing its possible mechanism of action. Materials and Methods: The MTT assay was used to investigate the inhibitory effect of artesunate on growth of breast cancer MCF-7 cells. For this aim, different concentrations of artesunate were used to treat the cells and flow cytometry assay was done followed by annexin V-FITC/PI staining. The activities of caspase-3, -8 and -9 were then determined by relative assay kits. Results: Based on the results from MTT assay, it was found that artesunate could significantly inhibit the growth of MCF-7 cells in a dose- and time-dependent manner. On the other hand, the flow cytometry findings showed that the anti-proliferative activity of artesunate on MCF-7 cells is due to apoptosis. Besides, caspase colorimetric assays revealed a significant rise in cellular levels of the initiators (caspase-8 and -9) and effector (caspase-3) in the cells treated by artesunate. Conclusions: According to our results, it could be concluded that artesunate could inhibit the growth of MCF-7 breast cancer cells through induction of apoptosis by intrinsic and extrinsic caspase-dependent pathways. Therefore, we claim that artesunate could be introduced as a suitable candidate for the treatment of the breast cancer.

In vitro inhibitory effect of artesunate on the vascular endothelial growth factor secreted by mononuclear cells in hand-foot-and-mouth disease children complicated with encephalitis

Objective To investigate the dynamics of white blood cell count (WBC), prealbumin (PA), high sensitive C-reactive protein (hs-CRP), and vascular endothelial growth factor (VEGF) in hand-foot-and-mouth disease (HFMD) complicated with or without encephalitis and in vitro inhibitory effects of artesunate on VEGF secretion of mononuclear cells from HFMD patients. Methods Peripheral blood mononuclear cells from healthy control group, HFMD group and HFMD combined with encephalitis group were isolated by Ficoll density gradient centrifugation and treated with different concentrations of artesunate (25, 50, 100 mg/L). The expression of VEGF in the supernatant was examined by ELISA double antibody sandwich method. The levels of WBC, PA and hs-CRP in the three groups were also detected. Multiple samples were compared by one-way analysis of variance. Multiple comparison was performed by Dunnett T3 test. Correlation of two variables was analyzed by Spearman correlation test. Results There were significant differences in the levels of WBC, hs-CRP, PA and VEGF between the HFMD combined with or without encephalitis group and health control group (F=172.69, 366.02, 166.32 and 5 941.89, respectively, all P<0.01). There were significant differences in the levels of VEGF secreted by mononuclear cells treated with three different concentrations of artesunate between the HFMD combined with or without encephalitis groups and health control group (F=194.265 and 4 750.69, respectively, both P<0.01). Correlation analysis showed that the VEGF level secretion by mononuclear cells from HFMD patients with or without encephalitis were both negatively correlated with different concentrations of artesunate (r=-0.903 and -0.969, both P<0.01). Conclusions Compared with HFMD without encephalitis, the secretion of VEGF by mononuclear cells, WBC and hs-CRP levels in HFMD complicated with encephalitis group all increase and PA decrease significantly. Artesunate can inhibit the secretion of VEGF by mononuclear cells of patients with hand-foot-and-mouth disease in a dose-dependent manner in vitro. Key words: Hand, foot and mouth disease; Encephalitis; Artesunate; Vascular endothelial growth factors

Artesunate Decreases β-Catenin Expression, Cell Proliferation and Apoptosis Resistance in the MG-63 Human Osteosarcoma Cell Line

Background: This study aims to determine the effects of artesunate on proliferation, apoptosis and β-catenin expression in the human osteosarcoma cell line MG-63. Methods: MG-63 cells in the logarithmic growth phase were collected and cultured with different concentrations of artesunate (12.5 µg/mL, 25 µg/mL and 50 µg/mL) for 24 h, 48 h and 72 h. The total number of MG-63 cells and the morphological changes were observed under an inverted microscope. The MTT assay was adopted to test the inhibition rate (IR) of cell growth. The apoptosis rate was detected using annexin V/propidium iodide (PI) staining. Cell cycle distribution was identified by flow cytometry (FCM), and the expression levels of β-catenin, cyclins and cyclin dependent kinases (CDKs) were measured using Western blotting. Results: The results of the MTT assay indicated that artesunate could remarkably inhibit MG-63 cell proliferation compared with the rates in the untreated control group (0 µg/mL artesunate), and the inhibitory effect was dose-dependent. The apoptosis rate of MG-63 cells was elevated as the concentration of artesunate increased, and all the rates were significantly higher than that in the control group. Additionally, as the artesunate concentration increased, the proportion of MG-63 cells in G0/G1 phase gradually declined whereas that of cells in the G2/M and S phases increased. Western blotting confirmed that a higher concentration of artesunate reduced the expression levels of β-catenin, cyclin A, cyclin D1 and CDK1 and increased the expression levels of cyclin B1; however, artesunate had no impact on CDK2 expression in MG-63 cells. Conclusion: These results demonstrated that artesunate can inhibit β-catenin expression and cell proliferation as well as promote cell apoptosis in MG-63 cells, which indicates that artesunate may serve as a promising drug in the clinical treatment of osteosarcoma.

Effect of Artesunate on Proliferation, Cell Cycle and Apoptosis of SKM-1 Cells and Its Underlying Mechanisms

To investigate the effects of artesunate (ART) on proliferation, cell cycle and apoptosis of SKM-1 cells in vitro and to explore the underlying mechanisms. After SKM-1 cells were treated with different concentrations of ART, the cell proliferation was determined by CCK-8 method. Apoptosis and distribution of cell cycle were detected by flow cytometry. Both DCFH-DA fluorescent probe and Fluo-3-Am fluorescent probe were used to detect the changes of intracellular reactive oxygen species (ROS) and calcium ion concentration. Western blot was used to measure the protein levels of BCL-2, BAX, BAD, P-BAD, survivin and XIAP. ART obviously inhibited the growth of SKM-1 cells in time and dose-dependent manners (r = -0.841; r = 0.-786). The antioxidant trolox-pretreatment significantly decreased the growth inhibition effect of ART on SKM-1 cells. Caspase inhibitor Ac-DEVD-CHO partially reduced the growth inhibition effect of ART on SKM-1 cells. After treatment with ART for 24 hours, the apoptosis of SKM-1 cells was found, the cell cycle of SKM-1 was arrested in G0/G1 phase, ART could elevate the levels of calciumion and reactive orygen. ART could significantly down-regulate the protein expression levels of P-BAD and survivin in SKM-1 cells, and showed a highly negative correlation with ART dose (r = -0.909; r = -0.849). On the contrary, ART had no significant effect on expression levels of BAD and XIAP in SKM-1 cells, and after ART treatment, although BCL-2 protein expression was not significantly different when compared with control group, but the BCL-2/BAX ratio significantly decreased and highly negatively correlated with ART dose (r = -0.866). The ART significantly suppresses the cell proliferation, induces the apoptosis and promoted cell cycle arrest at G0/G1 phase in SKM-1 cells. The mechanisms of ART anti-MDS is associated with the increase of intracellular calciumion concentration and ROS levels. In addition, the pro-apoptotic activity of ART may be involved in the regulation of BCL-2 /BAX ratio and the expressions of P-bad and survivin.

In vitro evaluation of the genotoxic and cytotoxic effects of artesunate, an antimalarial drug, in human lymphocytes

Artesunate is one of the main antimalarial drugs used in several countries. It is a semisynthetic compound derived from artemisinin, a substance extracted from the Chinese plant, Artemisia annua L. Despite the widespread use of artesunate as an antimalarial drug, there is a lack of data regarding its genotoxic effects in human lymphocytes. Therefore, in this study, we used the comet assay and micronucleus test to evaluate the possible genotoxic effects of artesunate in cultured human lymphocytes. In addition, cell death by necrosis and apoptosis was also assessed. Cells exposed to different concentrations of artesunate showed a significant concentration-dependent increase (P < 0.05) in DNA damage index and micronuclei frequency. A significant increase in the frequency of apoptotic and necrotic cells was also observed. Our results showed that artesunate is a genotoxic and cytotoxic compound in cultured human lymphocytes.

Artesunate induced morphological and mechanical changes of Jurkat cell studied by AFM

In this study, we have used atomic force microscopy (AFM) to study the morphology and mechanical property changes of Jurkat cells exposed to different concentrations of Artesunate (ART) for 24 h at single cellular level. Cell viability and proliferation assays were performed by using the Cell Counting Kit-8. The concentration of ART, which resulted in the inhibition rate >50% was selected. The AFM images revealed that the cell membrane changed and the ultrastructure also became complex. Mechanical properties of individual cell were tracked with AFM-based force spectroscopy. The force curves revealed that when a cell was exposed to the ART, the mechanical properties changed obviously. Treated cells had a lower adhesion force of 416.8+/-37.9 pN, whereas control group had a higher adhesion force of 1064.2+/-97.0 pN. The Young's modulus decreased to nearly one-third, from control group of 0.648+/-0.037 kPa to treated group of 0.254+/-0.035 kPa and the stiffness increased to nearly 1.5 times, from control group of 1.231+/-0.084 mN/m to treated group of 1.917+/-0.137 mN/m. These results suggest that ART can inhibit the proliferation of Jurkat and induce changes in the morphological structure and mechanical properties of Jurkat cells. The high resolution and high sensitivity of AFM can be used to detect morphological and mechanical properties of cells exposed to ART. The AFM may be developed to be a useful tool for detecting the cell death and evaluating the anti-carcinogen efficacy against tumor cell. SCANNING 31: 83-89, 2009. (c) 2009 Wiley Periodicals, Inc.